The present invention relates to a stabilized Tat antigen and to uses thereof for anti-HIV immunization.
Acquired immunodeficiency syndrome (AIDS) is a sexually transmissible disease caused by the human immunodeficiency virus (HIV type 1 (HIV-1) or type 2 (HIV-2)). This disease is constantly progressing and, at present, more than 42 million individuals are infected in the world. For this reason, the development of a vaccine is of the utmost urgency for combating this pandemic.
Numerous vaccine approaches have been developed for close to twenty years, without resulting in the development of an effective vaccine. However, the ever increasing knowledge of the infectious cycle and of the viral proteins responsible for progression to the AIDS stage has opened up the path for the use of novel promising vaccine targets: the HIV regulatory proteins. These proteins, which had originally been neglected, have for a few years been the subject of numerous studies due to their role in viral replication.
Among these proteins, the Tat transcriptional regulator of HIV represents a vaccine target which is particularly advantageous since the absence of progression to the AIDS stage is correlated with the presence of hightiter, anti-Tat antibodies and of specific cytotoxic cells (Zagury et al., J. Hum. Virol., 1998, 1: 282-292; Re et al., J. Clin. Virol., 2001, 21: 81-89; Van Baalen et al., J. Gen. Virol., 1997, 78: 1913-1918).
The Tat gene comprises 2 exons encoding a protein of 99 to 103 amino acids depending on the HIV strains. Exon 1, which encodes the first 72 amino acids, has a complete transactivation activity and comprises 5 domains: (1) the N-terminal domain (positions 1 to 21), which is important for the interaction with cell proteins, (2) the cysteine-rich domain (positions 22 to 37) containing 7 cysteine residues (positions 22, 25, 27, 30, 31, 34 and 37), among which 6 are strongly conserved, which domain is involved in transactivation, (3) the central (core) domain corresponding to positions 38 to 48, also involved in transactivation, (4) the basic domain (positions 49 to 57), which comprises the sequences involved in nuclear localization, transcellular transport and binding to the TAR (Trans-activation response) element of the viral LTR (Long Terminal Repeat), and which is also involved in the binding of Tat to heparin, and (5) the glutaminerich domain (positions 50 to 72). Exon 2, which is variable in size, encodes the C-terminal domain (positions 73 to the C-terminal end) which does not have transactivation activity but contains the RGD motif (arginine-glycine-aspartate; positions 78 to 80), required for Tat binding to integrin receptors.
In addition to the Tat protein of 99 to 103 amino acids, there also exists a truncated Tat protein of 86 amino acids, produced in vitro by generation of a stop codon at position 87, due to the mutation of HIV during passage in cell culture.
It has been shown that the isolated and purified, recombinant or synthetic Tat protein associates in solution so as to form oligomers (dimers and trimers) which are very stable, particularly resistant to denaturation and to reduction (100 mM DTT; Tosi et al., Eur. J. Immunol., 2000, 30: 1120-1126). Thus, Tat preparations are heterogeneous and comprise a mixture of monomers, dimers and trimers (patent application US 2003/0158134). However, the exact nature of the interactions involved in the formation of Tat oligomers is not known; the existence of disulfide bridges involving in particular the cysteine at position 37, or other strong chemical interactions, involving in particular polyvalent cations, preferably divalent cations (zinc, cadmium), has been suggested (Battaglia et al., Biochem. Biophys. Res. Commun., 1994, 201: 701-708; Frankel et al., Science, 1988, 240: 70-73; Huang et al., Biochem. Biophys. Res. Comm., 1996, 227: 615-621; Misumi et al., Aids Research and Human Retroviruses, 2004, 20: 297-304).
Tat is a transcription factor essential to viral replication (Fische et al., Nature, 1986, 320: 367-371), which can both activate latent HIV and deregulate the expression of other cellular genes, since it has the particularity of being released by the infected cells and incorporated into other infected or noninfected cells (transcellular transport: Ensoli at al., J. Virol., 1993, 67: 277-287; Chang at al., Aids, 997, 11: 1421-1431). It has been shown that Tat has toxic effects in vitro. These effects comprise: deregulation of cell signals involved in apoptosis, deregulation of the expression of parts of genes of the immune system, such as the interleukin 2 gene and the interferon-alpha gene, or genes encoding major histocompatibility complex (MHC) class I and class II molecules, and/or the induction of angiogenesis.
The relative role of the various forms of Tat in viral infection has not been elucidated and their role in antiviral immunity has not been studied. In fact, it would appear that Tat oligomers do not have any biological activity since, firstly, the extracellular Tat protein produced from mammalian cells is monomeric and, secondly, inhibition of the transactivating activity of Tat using antibodies, which specifically recognize the various forms of Tat, indicates that only the monomer is capable of entering cells and transactivating the viral LTR (Rice at al., Virology, 1991, 185: 451-454; Tosi et al., mentioned above).
Thus, Tat has numerous biological activities and could play a role both in the viral dissemination and in the pathogenesis: progression to the AIDS stage and AIDS-related pathologies, such as Kaposi's sarcoma.
Consequently, although a nonmodified, biologically active Tat protein is capable of protecting monkeys against HIV-1 infection (Cafaro et al., Nat. Med., 1999, 5: 643-650), the use of a toxic protein cannot be envisaged as an immunogen for human immunization.
For this reason, various approaches have been envisaged for obtaining biologically inactive Tat derivatives which can be used as a vaccine in humans.
The inactivation is mainly related to the elimination of the ability of Tat to transactivate transcription of the viral genome, and then to other activities, such as inhibition of the suppression of T cell proliferation. These studies have resulted in the discovery of biologically inactive derivatives of Tat, obtained by means of:                mutations in the sequence of the Tat gene: (i) deletions of the —NH2 or —COOH ends, or else deletion or substitution of the cysteine residues (international application WO 95/31999), (ii) conservative substitution of all the cysteine residues of the cysteine-rich domain (cysteine→serine; international application WO 03/054006), (iii) substitution of at least two amino acid residues of positions 49 to 72 and/or 73 to 101, in particular of positions 49 to 57 (K51T, R52L, R55L, R57L), of the RGD domain (G79A) or of positions 88 to (K89L, E92Q), and optionally of the cysteine at position 27 (C27S) (international application WO 03/057885), and (iv) deletion of an amino acid residue (C22, T23, N24, Y26, K28/29, C30, C31, F32, K33, E35, F38, K41, Y47, A57) or substitution of an amino acid residue (T23A, N24A, C22G, K41T; international application WO 99/27958),        analysis of the Tat gene mutations naturally present in nonprogressive seropositive individuals: variants of the N-terminal region or of exon 2 (international application WO 01/12220); variant derived from the Oyi isolate, having the substitution C22S (international application WO 00/61067),        chemical or physical treatments of the Tat protein: (i) treatment with an aldehyde, such as formaldehyde and glutaraldehyde (PCT international application WO 96/27389), (ii) carboxymethylation of cysteines by means of iodoacetic acid or of iodoacetamide (PCT international application WO 99/33872), and (iii) oxidation, in particular with hydrogen peroxide or sodium periodate or else irradiation (patent application US 2003/0215797; international application WO 01/12220; Cohen et al., P.N.A.S., 1999, 96: 10842-10847), and        purification of the recombinant Tat protein by means of a method for eliminating the contaminating RNA and endotoxin (PCT international application WO 03/073984).        
It has in particular been shown that a Tat derivative in which cysteines 22 and 37 are substituted with serines is immunogenic (Caselli et al., J. Immunol., 1999, 162: 5631-5638) and that a carboxymethyl derivative of Tat is immunogenic and capable of inducing a slowing down of the progression of the disease in monkeys (Pauza et al., P.N.A.S., 2000, 97, 3515-3519).
These results indicate that a Tat immunogene which is biologically active or has been inactivated beforehand can contribute to the setting up an effective HIV vaccine. However, the effectiveness of a vaccine depends to a large extent on its ability to induce a strong immune response. Now, the Tat molecule is known for its immunosuppressive capacities (Cohen et al., mentioned above) and is weakly immunogenic in animals, in particular in monkeys. Thus, the slowing down of the disease induced by the carboxymethylated Tat toxoid requires a series of 5 immunizations (Pauza et al., P.N.A.S., 2000, 97: 3515-3519) and the protection provided by the biologically active Tat molecule has been obtained after a series of 9 or 10 immunizations, according to protocols (Cafaro et al., mentioned above).
Consequently, in order to develop a vaccine which is effective against HIV, there exists a real need to develop a Tat-derived antigen which exhibits increased immunogenicity compared with that of the Tat antigens of the prior art.
The inventors have given themselves the aim of developing such an antigen.